Inflammation is an essential protective mechanism that must be tightly regulated to ensure that responses are of a suitable magnitude and duration. Dysregulated inflammation underlies chronic diseases (e.g. inflammatory bowel disease). This proposal tests the general hypothesis that proteases from inflammatory and epithelial cells of the inflamed colon signal to the epithelium by activating receptors and ion channels, including protease-activated receptor 2 (PAR2), transient receptor potential vanilloid 4 (TRPV4) and the epithelial sodium channel (ENaC). The scaffolding protein 2-arrestin (2arr) assembles receptor and channel signalosomes that define the subcellular location, duration and outcome of inflammatory signaling. Intracellular proteases that degrade or deubiquitinate activated receptors, channels and 2arrs destabilize signalosomes and terminate inflammatory signaling. Approaches will include studies of human colonocyte cell lines, isolated human and mouse tissues, and mice with tissue-specific and inducible deletion of proteases, PAR2, TRPV4 and 2arrs. Aim 1 tests the hypothesis that proteases induce assembly of PAR272arr2 signalosomes that convey sustained inflammatory signals within the endosomal network. Experiments determine whether: 1) endosomal signalosomes transmit sustained proinflammatory signals;2) endosomal proteases that degrade PAR2 or deubiquitinate 2arr2 destabilize signalosomes and terminate proinflammatory signals;and 3) PAR2 and 2arr2 mediate the proinflammatory actions of proteases in the colon. Aim 2 tests the hypothesis that proteases activate plasma membrane ion channels indirectly via PAR2 and directly by channel proteolysis, to control inflammatory signaling and electrolyte transport. Experiments determine whether: 1) PAR272arr17TRPV4 signalosomes at the basolateral plasma membrane mediate cooperative proinflammatory signaling of PAR2 and TRPV4;2) TRPV4 and 2arr1 mediate the proinflammatory actions of proteases in the colon;and 3) luminal proteases cleave ENaC at the apical plasma membrane and stimulate Na+ and fluid transport that is protective in colitis. Aim 3 tests the hypothesis that proteases that are generated during inflammation activate receptors and channels on epithelial cells and play a causative role in inflammatory disease. Since proteases are regulated by post-translational control of activity, activity-based probes that interact only with active proteases will be used to identify and localize active proteases in diseased tissues. Experiments will: 1) identify and localize proteases that are activated during the initiation, consolidation and resolution of colitis in mice;2) identify and localize proteases that are activated in mucosal biopsies from patients with colitis;and 3) determine the causative role of proteases, PAR2, TRPV4 and 2arrs in colitis in mice. The proposal will identify new mechanisms of protease-regulated inflammation, with implications for improved and more specific therapy, and non-invasive detection of inflammation prior to irreversible damage. The results will provide insights into inflammatory bowel disease that are applicable to inflammatory diseases that affect all systems. PUBLIC HEALTH RELEVANCE: Chronic inflammation underlies diseases that affect all organ systems that are a major cause of morbidity and mortality. Difficulties in effective treatment of inflammatory diseases are related to an incomplete understanding of the mechanisms that transmit inflammatory signals, and to inadequate approaches for the early detection of disease prior to irreversible organ damage. This proposal seeks to determine the role of proteases in chronic inflammation of the intestine. Studies will define the mechanisms and causative role of proteases in signaling inflammation, and pioneer new approaches for the early detection of inflammation by non-invasive approaches prior to irreversible organ damage. The proposal will provide new information that will facilitate the treatment, detection and diagnosis of inflammatory diseases that are a global cause of morbidity and mortality.